Browsing by Subject "proteomics"

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    Detection of Alternative Splice Variants at the Proteome Level in Aspergillus flavus
    (2010) Chang, Kung-Yen; Georgianna, D Ryan; Heber, Steffen; Payne, Gary A; Muddiman, David C
    Identification of proteins from proteolytic peptides or intact proteins plays an essential role in proteomics. Researchers use search engines to match the acquired peptide sequences to the target proteins. However, search engines depend on protein databases to provide candidates for consideration. Alternative splicing (AS), the mechanism where the exon of pre-mRNAs can be spliced and rearranged to generate distinct mRNA and therefore protein variants, enable higher eukaryotic organisms, with only a limited number of genes, to have the requisite complexity and diversity at the proteome level. Multiple alternative isoforms from one gene often share common segments of sequences. However, many protein databases only include a limited number of isoforms to keep minimal redundancy. As a result, the database search might not identify a target protein even with high quality tandem MS data and accurate intact precursor ion mass. We computationally predicted an exhaustive list of putative isoforms of Aspergillus flavus proteins from 20 371 expressed sequence tags to investigate whether an alternative splicing protein database can assign a greater proportion of mass spectrometry data. The newly constructed AS database provided 9807 new alternatively spliced variants in addition to 12 832 previously annotated proteins. The searches of the existing tandem MS spectra data set using the AS database identified 29 new proteins encoded by 26 genes. Nine fungal genes appeared to have multiple protein isoforms. In addition to the discovery of splice variants, AS database also showed potential to improve genome annotation. In summary, the introduction of an alternative splicing database helps identify more proteins and unveils more information about a proteome.
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    Elucidating the Molecular Composition of Cartilage by Proteomics.
    (J Proteome Res, 2016-02-05) Hsueh, Ming-Feng; Khabut, Areej; Kjellström, Sven; Önnerfjord, Patrik; Kraus, Virginia Byers
    Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four-times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate, and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic), and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of osteoarthritis (OA), OA progression, and the joint specific biomarkers.
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    ItemOpen Access
    Glucocorticoids Preferentially Influence Expression of Nucleoskeletal Actin Network and Cell Adhesive Proteins in Human Trabecular Meshwork Cells.
    (Frontiers in cell and developmental biology, 2022-01) Bachman, William; Maddala, Rupalatha; Chakraborty, Ayon; Eldawy, Camelia; Skiba, Nikolai P; Rao, Ponugoti V
    Clinical use of glucocorticoids is associated with increased intraocular pressure (IOP), a major risk factor for glaucoma. Glucocorticoids have been reported to induce changes in actin cytoskeletal organization, cell adhesion, extracellular matrix, fibrogenic activity, and mechanical properties of trabecular meshwork (TM) tissue, which plays a crucial role in aqueous humor dynamics and IOP homeostasis. However, we have a limited understanding of the molecular underpinnings regulating these myriad processes in TM cells. To understand how proteins, including cytoskeletal and cell adhesion proteins that are recognized to shuttle between the cytosolic and nuclear regions, influence gene expression and other cellular activities, we used proteomic analysis to characterize the nuclear protein fraction of dexamethasone (Dex) treated human TM cells. Treatment of human TM cells with Dex for 1, 5, or 7 days led to consistent increases (by ≥ two-fold) in the levels of various actin cytoskeletal regulatory, cell adhesive, and vesicle trafficking proteins. Increases (≥two-fold) were also observed in levels of Wnt signaling regulator (glypican-4), actin-binding chromatin modulator (BRG1) and nuclear actin filament depolymerizing protein (MICAL2; microtubule-associated monooxygenase, calponin and LIM domain containing), together with a decrease in tissue plasminogen activator. These changes were independently further confirmed by immunoblotting analysis. Interestingly, deficiency of BRG1 expression blunted the Dex-induced increases in the levels of some of these proteins in TM cells. In summary, these findings indicate that the widely recognized changes in actin cytoskeletal and cell adhesive attributes of TM cells by glucocorticoids involve actin regulated BRG1 chromatin remodeling, nuclear MICAL2, and glypican-4 regulated Wnt signaling upstream of the serum response factor/myocardin controlled transcriptional activity.